Bone tissue undergoes constant remodeling to fulfill its principal functions of mechanical support, maintenance of calcium homeostasis, and as a stem cell supplier. This process is mediated by two cell lineages: the hematopoietic bone-resorbing osteoclasts and the bone-forming mesenchymal osteoblasts and osteocytes. Under physiological conditions, the balance between bone formation and resorption is tightly regulated and determines bone density. This balance is based on the RANK/RANKL/OPG pathway.
Receptor activator of nuclear factor-κB (RANK) is a member of the tumor necrosis factor family expressed by osteoclasts and their precursors. The interaction of RANK with RANKL (its ligand) has been identified as the final common pathway through which bone resorption is regulated. By binding to its receptor RANK on osteoclastic precursors, RANKL controls the differentiation, proliferation, and survival of osteoclasts. Osteoprotegerin (OPG) is the natural inhibitor of RANKL. RANK and RANKL are expressed in many regular cell types (Theoleyre et al. 2004) but their activity is the most prominent in bone tissue, skin (Duheron et al. 2011) and mammary glands (Gonzalez-Suarez et al 2010; Schramek et al 2010). RANK-RANKL expression is severely enhanced in non-bone cancer cells such as breast or melanoma cancers, in their associated metastasis (Jones et al. 2006) and in primary bone cancers as in osteosarcoma (Mori et al. 2007).
Disruption of the homeostatic balance can lead to pathologic bone loss, such as in age-related osteoporosis, periodontal disease or inflammatory rheumatoid arthritis, or to excessive bone formation, such as in skeletal malformations linked or not with genetic mutations and/or polymorphisms (Whyte et al. 2009) or to alteration in bone remodeling. Perturbations in the ratio of OPG to RANKL have been demonstrated to occur with estrogen deficiency, hyperparathyroidism, and other disorders that stimulate bone resorption. RANKL is also expressed by lymphocytes and synovial fibroblasts and may mediate bone loss associated with inflammatory conditions.
The discovery of the RANK/RANKL/OPG pathway and its implications in the pathogenesis of bone diseases provided a molecular target for therapies to improve bone health.
The development of small molecules, OPG mimetics which target RANKL have been suggested for the development of therapeutic agents to treat bone diseases, in particular bone resorptive diseases. Cheng and his co-workers (Cheng et al. 2004) have designed such peptides derived from OPG to block RANKL. Their most promising peptide OP3-4 was able to directly bind RANKL with a measurable interaction by surface plasmon resonance. This interaction was sufficient to reduce in vitro osteoclastogenesis and protect mice in vivo from bone loss.
Another approach initiated by Takasaki in 1997 (Takasaki et al. 1997) was to design bio-compatible molecules able to target RANKL based on peptides derived from the RANK sequence. Their work, initially targeting the TNF-alpha/TNFR interaction, proved useful to block the RANKL/RANK interaction in a TNF-independent way (Aoki et al. 2006). Their most effective peptide named WP9QY was able to inhibit in vitro osteoclastogenesis in a dose-dependent manner and prevent in vivo bone loss in a mouse osteoporosis-induced model.
These peptides were derived from the native sequence of the partner they were targeting, based on assumptions made upon models based on other members of the TNF/TNF-R family: i.e. OP3-4 was selected from the putative OPG-RANKL interface (the model for OPG used TNFR, Fas and TRAIL crystallographic structures) and WP9QY was selected from the putative RANKL-RANK interface (models were based on the TNF-R/TNF-β crystallographic structure). Although their biological activity showed promising results in in vitro and in vivo models, their therapeutic interest was potentially limited by their relatively low binding affinity to RANKL, in comparison to the binding of RANKL to RANK.
Over the past decade, there have been tremendous advances in the management of metabolic bone disorders with the introduction of novel bone-chelating agents (zoledronate being the most prominent treatment to date) and the development of a monoclonal antibody (denosumab) directed against RANKL (Baron et al. 2011). Although these therapies show promising improvements in the treatment of bone diseases, in particular in bone resorptive diseases, their application is limited by the poor bioavailability and/or stability of large macromolecules, such as antibodies or chimeras, the mode of administration, the high cost and the risk of mild to severe and sometimes even life-compromising side effects, such as skin rashes, immunogenicity, osteonecrosis of the jaw or increased trabecular bone mineral density leading to growth retardation.
There remains, therefore, a significant need for new and improved compounds for the prevention and/or treatment of bone diseases, in particular of bone resorptive diseases, which are effective in inhibiting osteoclastogenesis, cheap to produce, possess a high bioavailability, which may be easily administered to patients while being without severe side effects. The present inventors have made a significant step forward with the invention disclosed herein.